Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), represents a severe world health crisis (4,000 TB deaths daily). A better understanding of Mtb biology and pathogenesis will drive development of novel strategies to control the TB epidemic. In bacterial pathogens, like Mtb, many proteins are exported to the bacterial cell envelope or the extracellular environment in order to carry out critical functions in bacterial physiology, virulence or immune responses. Thus, Mtb pathways that export proteins could be targeted or exploited for the development of novel anti-TB control measures. However, significant gaps exist in our understanding of Mtb protein export. This proposal is to determine the mechanism of SatS, a novel protein export chaperone we discovered in Mtb. SatS is a chaperone for a subset of proteins that are exported by the specialized SecA2 protein export pathway, and SatS is required for intracellular growth of Mtb in macrophages. However, SatS shares no sequence or structural similarity with any previously characterized proteins and the mechanism of SatS is completely unknown. Substrate specificity and the scope of SatS substrates are also unknown. Aim 1 of this proposal is to determine the mechanistic details of SatS function in protein export. Aim 2 is to determine the structures of SatS and that of SatS in complex with peptides of substrates. Aim 3 is to identify SatS-binding sites in substrates and to identify additional SatS substrates. By completing these Aims, we will determine the mechanism and contribution of SatS to Mtb protein export and biology. More broadly, these studies will improve our understanding of bacterial strategies for exporting proteins and of the diversity of molecular chaperones across biological systems. Long-term, the results could lead to strategies for inhibiting protein export as a novel anti-TB therapy. Alternatively, the knowledge gained could be harnessed to engineer mycobacterial strains with improved capacity to export proteins for use as experimental tools or as live, attenuated vaccines with enhanced antigen export.